P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells

Tumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.Subject terms: Ligand-gated ion channels, Metastasis, Melanoma, Ion channel signalling, miRNAs     

Biochemical and immunochemical characterization of hop-hornbeam (Ostrya Carpinifolia Scop.) pollen

In the last few years Ostrya carpinifolia pollen is consideredas an important cause of respiratoryallergy in Mediterranean areas. The concentration ofthe pollen was measured over a period of fifteen yearsfrom 1981 to 1996 in an area around Genoa; the resultsof this study have clearly indicated an increasingtrend that correlate with persons sensitization.In this study we sought to define the immunochemical andbiochemical properties of hop-hornbean pollen. Soluble proteins extracted from Ostryacarpinifolia pollen and from the taxonomicallyrelated species Corylus Avellana, were analyzedby polyacrylamide gel electrophoresis (SDS-PAGE), byhorizontal isoelectrofocusing (IEF) and by twodimensions electrophoresis (2D-PAGE). Allergenicproteins were identified with sera of sensibilizedpatients and cross-reactivity was evaluated byimmunoblotting techniques. The electrophoreticanalysis showed a partial identity between theproteins from Ostrya and Corylus extracts. The immunoblotting assay, developed withhuman IgE from subjects allergic to hop-hornbeampollen, displayed the major IgE reactivity for acomponent with a molecular weight of 17 kDa expressedin both Ostrya and Corylus extracts. This reactivity is consistent with the presence ofBet v 1 that is described as the major pollen allergenin the Betulaceae and Corylaceae families. Sera fromsubjects allergic to Ostrya were then preadsorbed with recombinant Bet v 1 immobilized in the Pharmacia CAP System; a significant reduction ofthe IgE binding activity was observed after thetreatment. We therefore suggest that Bet v 1 couldbe one of the allergenic proteins present in theOstrya pollen possibly being responsible forcross-reactivity with other members of taxonomicallyrelated families.     

CD39 is highly involved in mediating the suppression activity of tumor-infiltrating CD8+ T regulatory lymphocytes

CD39 is an ectoenzyme, present on different immune cell subsets, which mediates immunosuppressive functions catalyzing ATP degradation. It is not known whether CD39 is expressed and implicated in the activity of CD8+ regulatory T lymphocytes (Treg). In this study, CD39 expression and function was analyzed in both CD8+ and CD4+CD25^hi Treg from the peripheral blood of healthy donors as well as from tumor specimens. CD39 was found expressed by both CD8+ (from the majority of healthy donors and tumor patients) and CD4+CD25^hi Treg, and CD39 expression correlated with suppression activity mediated by CD8+ Treg. Importantly, CD39 counteraction remarkably inhibited the suppression activity of CD8+ Treg (both from peripheral blood and tumor microenvironment) suggesting that CD39-mediated inhibition constitutes a prevalent hallmark of their function. Collectively, these findings, unveiling a new mechanism of action for CD8+ Treg, provide new knowledge on intratumoral molecular pathways related to tumor immune escape, which could be exploited in the future for designing new biological tools for anticancer immune intervention.     

Break zones in the distributions of alleles and species in alpine plants

Aim We test for the congruence between allele‐based range boundaries (break zones) in silicicolous alpine plants and species‐based break zones in the silicicolous flora of the European Alps. We also ask whether such break zones coincide with areas of large elevational variation. Location The European Alps. Methods On a regular grid laid across the entire Alps, we determined areas of allele‐ and species‐based break zones using respective clustering algorithms, identifying discontinuities in cluster distributions (breaks), and quantifying integrated break densities (break zones). Discontinuities were identified based on the intra‐specific genetic variation of 12 species and on the floristic distribution data from 239 species, respectively. Coincidence between the two types of break zones was tested using Spearman’s correlation. Break zone densities were also regressed on topographical complexity to test for the effect of elevational variation. Results We found that two main break zones in the distribution of alleles and species were significantly correlated. Furthermore, we show that these break zones are in topographically complex regions, characterized by massive elevational ranges owing to high mountains and deep glacial valleys. We detected a third break zone in the distribution of species in the eastern Alps, which is not correlated with topographic complexity, and which is also not evident from allelic distribution patterns. Species with the potential for long‐distance dispersal tended to show larger distribution ranges than short‐distance dispersers. Main conclusions We suggest that the history of Pleistocene glaciations is the main driver of the congruence between allele‐based and species‐based distribution patterns, because occurrences of both species and alleles were subject to the same processes (such as extinction, migration and drift) that shaped the distributions of species and genetic lineages. Large elevational ranges have had a profound effect as a dispersal barrier for alleles during post‐glacial immigration. Because plant species, unlike alleles, cannot spread via pollen but only via seed, and thus disperse less effectively, we conclude that species break zones are maintained over longer time spans and reflect more ancient patterns than allele break zones.     

Kinetics of fluid flux in the rat diaphragmatic submesothelial lymphatic lacunae

The specific role of loops and/or linear segments in pleural diaphragmatic submesothelial lymphatics was investigated in seven anesthetized, paralyzed, and mechanically ventilated rats. Lymphatic loops lay peripherally above the diaphragmatic muscular plane, whereas linear vessels run over both the muscular and central tendineous regions. Lymph vessel diameter, measured by automatic software analysis, was significantly greater (P < 0.01) in linear vessels [103.4 +/- 8.5 microm (mean +/- SE), n = 18] than in loops (54.6 +/- 3.3 microm, n = 21). Conversely, the geometric mean of intraluminal flow velocity, obtained from the speed of distribution of a bolus of fluorescent dextrans injected into the vessel, was lower (P < 0.01) in linear vessels (26.3 +/- 1.4 microm/s) compared with loops (51.3 +/- 3.2 microm/s). Lymph flow, calculated as the product of flow velocity by vessel cross-sectional area, was similar in linear vessels and in individual vessels of a loop, averaging 8.6 +/- 1.6 nl/min. Flow was always directed from the diaphragm periphery toward the medial tendineous region in linear vessels, whereas it was more complex and evidently controlled by intraluminal unidirectional valves in loops. The results suggest that loops might be the preferential site of lymph formation, whereas linear vessels would be mainly involved in the progression of newly formed lymph toward deeper collecting diaphragmatic ducts. Within the same hierarchic order of diaphragmatic lymphatic vessels, the spatial organization and geometrical arrangement of the submesothelial lacunae seem to be finalized at exploiting the alternate contraction/relaxation phases of diaphragmatic muscle fibers to optimize fluid removal from serosal cavities.     

Low intraspecific aggressiveness in two obligate plant-ant species

Little is known about the aggressiveness of plant-ants typically living in isolated trees nor about how that aggressiveness varies based on this isolation. Here, we examine intra- and interspecific aggressiveness between workers of two Allomerus species associated with two different myrmecophytes. In both cases, the level of intraspecific aggressiveness is very low whatever the distance separating the tested nests, while interspecific conflicts are always violent. Similar patterns of aggressiveness have been reported in various ant species, but the strictly arboreal life of Allomerus ants associated with the isolation of their adult colonies highlight different ecological conditions that might explain the lack of aggressiveness between conspecifics. Received 18 December 2007; revised 12 March 2008; accepted 13 March 2008.